Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides.

Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene. Both intermediates were further metabolized to unknown substances. The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry.     

Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides.

Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene. Both intermediates were further metabolized to unknown substances. The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary excretion of isomers of biliverdin after destruction in vivo of haemoproteins and haemin.

The amount and isomeric composition of urinary biliverdin in rabbits were analysed by h.p.l.c. Physiological values were maintained after the injection of haemin. On the other hand, when haemoglobins from several mammalian species were injected into rabbits, the excretion of biliverdin-IX alpha and biliverdin-IX beta were increased 6-18-fold and 32-66-fold respectively over physiological excretion. Injection of myoglobin resulted in a 44-fold increase in excretion of the IX alpha-isomer. Coupled oxidation with ascorbate of haemoglobin and myoglobin by oxygen produced mainly the IX alpha- and IX beta-isomers from haemoglobin and the IX alpha-isomer from myoglobin. The destruction of part of the haem from injected haemoproteins by non-enzymic chemical degradation would account for the observed respective increases in the excretion of biliverdin isomers. The excretion of biliverdin isomers after the injection of phenylhydrazine into rabbits was similar to that after the injection of haemoglobin.     

Synthesis of 2-deoxy-beta-D-ribose 1-phosphate, NMR comparison and its enzymatic activity for structural elucidation of synthetic alpha-isomer

2-Deoxy-beta-D-ribose 1-phosphate (1) was synthesized in a stereoselective manner and isolated with no detectable contamination by its alpha-isomer (4). Explicit configuration of 4 was first determined by NMR comparison with 1 judging from NOE results and their coupling constants. Natural purine nucleoside phosphorylase (PNPase) did not recognize 1 and gave no products such as alpha- or beta-deoxynucleosides.     

Stereoselective formation of a cholesterol ester conjugate from fenvalerate by mouse microsomal carboxyesterase(s)

In accordance with in vivo findings, of the four chiral isomers of fenvalerate (S-5602 Sumicidin, Pydrin, [RS]-alpha-cyano-3-phenoxybenzyl [RS]-2-(4-chlorophenyl)isovalerate), only the [2R, alpha S]-isomer (B-isomer) yielded cholesteryl [2R]-2-(4-chlorophenyl)isovalerate (CPIA-cholesterol ester) in the in vitro study using several tissue homogenates of mice, rats, dogs, and monkeys. There were species differences in the extent of CPIA-cholesterol-ester formation, with mouse tissues showing relatively higher activity than those of other animals. The kidney, brain, and spleen of mice showed relatively higher capacities to form this ester compared to other tissues, and the enzyme activity was mainly localized in microsomal fractions. The CPIA-cholesterol ester did not seem to be produced by three known biosynthetic pathways of endogenous cholesterol esters--acyl-CoA:cholesterol O-acyltransferase (ACAT), lecithin:cholesterol O-acyltransferase (LCAT), and cholesterol esterase. Carboxyesterase(s) of mouse kidney microsomes solubilized by digitonin hydrolyzed only the B alpha-isomer of fenvalerate, yielding CPIA, whereas they yielded the corresponding cholesterol ester in the presence of artificial liposomes containing cholesterol. Thus, it appears that the stereoselective formation of the CPIA-cholesterol ester results from the stereoselective formation of the CPIA-carboxyesterase complex only from the B alpha-isomer, which subsequently undergoes cleavage by cholesterol to yield the CPIA-cholesterol ester.     

Bovine complex I is a complex of 45 different subunits

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Ibeta, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.     

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by B?hmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.     

Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.     

An enzyme-linked immunoassay for lipoprotein lipase

Polyclonal antibodies against bovine milk lipoprotein lipase (LPL) were used to generate an enzyme-linked immunosorbent assay (ELISA) for rat LPL. The antibodies to LPL were affinity purified on bovine LPL columns and were shown to be specific for LPL by immunoprecipitation and enzyme inhibition. The solid-phase ELISA was sensitive from 1.0 to 20 ng/ml of LPL and paralleled enzyme activity. Denatured rat LPL showed the same LPL mass as undenatured samples, allowing LPL mass to be quantitated effectively in a variety of rat tissue extracts.     

Expression of bovine F1-ATPase with functional complementation in yeast Saccharomyces cerevisiae

The mitochondrial F(1)F(0)-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of approximately 600 kDa. F(1)-ATPase is composed of alpha(3)beta(3)gammadeltaepsilon with an overall molecular mass of 370 kDa. The genes encoding bovine F(1)-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (DeltaalphaDeltabetaDeltagammaDeltadeltaDeltaepsilon). This strain expressing bovine F(1) is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F(1)-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F(1)- or F(1)F(0)-ATP synthase.     

Bradykinin and kininogens in bovine milk

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z 1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation. In apparent contrast with bovine plasma kininogens, the forms of kininogen which occur in milk include a high Mr kininogen (Mr greater than 68,000) and a low Mr kininogen (Mr 16,000-17,000). Kinin formation from the high Mr kininogen is catalyzed by porcine pancreatic kallikrein or trypsin.     

Adenine N3 is a main alkylation site of styrene oxide in double-stranded DNA

Styrene 7,8-oxide (SO), a major metabolite of styrene, is classified as a probable human carcinogen. In the present work, salmon testis DNA was reacted with SO and the alkylation products were analysed after sequential depurination in neutral or acidic conditions followed by HPLC separation and UV-detection. A novel finding was that the N-3 position of adenine was the next most reactive alkylation site in double-stranded DNA, comprising 4% of the total alkylation, as compared to alkylation at the N-7 position of guanine, 93% of the total alkylation. Both alpha- and beta-products of SO were formed at these two sites. Other modified sites were N2-guanine (1.5%, alpha-isomer), 1-adenine (0.4%, both isomers) and N6-adenine (0.7%, both isomers) as well as 1-hypoxanthine (0.1%, alpha-isomer), formed by deamination of the corresponding 1-adenine adduct. The results indicated that in double-stranded DNA N-7 of guanine and N-3 of adenine account for 97% of alkylation by SO. However, these abundant adducts are not stable, the half-life of depurination in DNA for 3-substituted adenines being approximately 10 and approximately 20 h, for alpha- and beta-isomers, respectively, and 51 h for both isomers of 7-substituted guanines.     

Characterization and synthesis of mono- and diphytanyl ethers of glycerol

The methanolyzed lipids of the extreme halophile, Halobacterium cutirubrum, were separated into glycerol diether and glycerol monoether fractions. The diether was shown by synthesis to be 2,3-di-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl)-sn-glycerol. The monoether fraction was separated by thin-layer chromatography on boric acid-impregnated silicic acid into about equal amounts of alpha- and -isomers. The alpha-isomer was found to be identical with the synthetic 3-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl)-sn-glycerol, and the -isomer was identical with the synthetic 2-O-(3'R,7'R,11'R,15'-tetramethylhexadecyl) glycerol.     

Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).     

Identification of the human beta-casein C-terminal fragments that specifically bind to purified antibodies to bovine beta-lactoglobulin

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine beta-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine beta-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine beta-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass >or= 1,000 Da cross-reactive with antibodies to bovine beta-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine beta-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human beta-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine beta-LG and allows the identification of possible common epitopes between the two proteins. No bovine beta-LG peptides with a molecular mass >or= 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.